Stem cells have the ability to differentiate into a variety of cells and tissues. Thus, considerable attention has focused on stem cells and their uses in a multitude of applications, including tissue engineering, tissue regeneration, and gene delivery. Stem cells have been isolated from both embryonic and adult tissues. Somatic stem cells that are derived from adult tissue still have the ability to renew adult tissues (Fuchs and Segre, 2000). Thus, in light of the ongoing controversies surrounding the use of embryonic stem cells, the use of somatic stem cells are a particularly attractive alternative.
The presence of stem cells in somatic tissues has been well established using functional tissue cell transplantation assays (Reisner et al., 1978). However, isolation and propagation of somatic stem cells has proven difficult. Methods to isolate and expand stem cells from somatic tissue, particularly without significant differentiation, are highly desirable. There have been some questions raised regarding how multi-potent adult stem cells are related to embryonic stem cells. Thus, it is important to be able to obtain and cultivate many different types of somatic stem cells. In particular, the availability of a method to propagate liver stem cells from adult tissues would greatly contribute to the treatment of chronic and acute liver failure, the identification of liver stem cell markers, as well as tissue engineering, e.g. generation of artificial liver.
There has been considerable difficulty encountered in obtaining human somatic liver stem cells that can be propagated and cultured ex vivo. One factor is the predominant way somatic stem cells divide is by asymmetric cell kinetics. During asymmetric kinetics, one daughter cell divides with the same kinetics as its stem cell parent, while the second daughter gives rise to a differentiating non-dividing cell lineage. The second daughter may differentiate immediately; or, depending on the tissue, it may undergo a finite number of successive symmetric divisions to give rise to a larger pool of differentiating cells.
Such asymmetric cell kinetics are a major obstacle to somatic cell expansion in vitro (Merok and Sherley, 2001; Rambhatla et al., 2001; Sherley, 2002). In culture, continued asymmetric cell kinetics results in dilution and loss of an initial relatively fixed number of stem cells by the accumulation of much greater numbers of their terminally differentiating progeny. If a sample includes both exponentially growing cells as well as somatic stem cells, the growth of the exponentially growing cells will rapidly overwhelm the somatic stem cells, leading to their dilution. Even in instances where it is possible to select for relatively purer populations, for example by cell sorting, asymmetric cell kinetics prevent expansion.
Furthermore, previous investigative methods for expanding post-natal human liver cells have required the use of feeder layers and biological matrices to maintain the cells in culture. With such methods, post-natal human liver cells could only be maintained for a few passages.
Thus, despite the need for methods to isolate liver stem cells from an individual and expand them ex vivo, it has not been possible to do so.